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Mandibular Explant Assay for Investigating Extrinsic Stimuli on Bone and Cartilage Development
研究骨及软骨外源刺激的下颌外植体实验   

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Abstract

A major issue in developmental biology is to determine how embryonic tissues respond to molecular signals in a timely manner and given the position-restricted instructions during morphogenesis, of which Meckel’s cartilage is a classical example. The ex-vivo explant model is a practical and convenient system that allows investigation of bone and cartilage responses to specific stimuli under a controlled manner that closely mimics the in vivo conditions. In this protocol, the explant model was applied to test whether Meckel’s cartilage and surrounding tissues are responsive to the Endothelin1 (Edn1) signaling molecule and whether it would rescue the phenotype of genetic mutations. The system allows a high degree of manipulation in terms of the concentrations of exogenous compounds added to the explant, time points with regards to measuring mandibular development, and the method of application of exogenous molecules and teratogens.

Keywords: Ex-vivo mandibular explant(下颌的体外分离), Craniofacial development(颅颌面发育), Mandibular dysplasia(下颌发育不良), Mandibular hypoplasia(下颌发育不全), Agnathia(无颌畸形), Mandibular patterning(下颌形态), Meckel’s cartilage(麦克尔软骨), Embryonic development(胚胎发育)

Background

Craniofacial malformations are among the most frequent congenital birth defects in humans (Miettinen et al., 1999). Many of these malformations occur during facial morphogenesis, a complex multi-step process in which cranial neural crest cells migrate to pharyngeal arches to give rise to many facial structures (Jin et al., 2011).

Both variations within specific genes as well as gene-gene interactions can lead to craniofacial deformations. Mutations in IRF6, a gene that contributes to the formation of ectoderm and epithelium in the head and face, can result in cleft lip, cleft palate, and mandibular abnormalities. In comparison to IRF6, the TWIST1 gene regulates neural tube closure during embryonic development and cranial suture fusion during skull development. Mutations in TWIST1 can cause craniosynostosis, mandibular hypoplasia, and cleft palate (Fakhouri et al., 2017). Inhibition or alteration of IRF6 and TWIST1 expression can be done similar to the methods performed by Miettinen et al. (1999) with EGF receptors in order to examine their roles in craniofacial development. However, difficulties arise in in vivo experiments when the study begins to incorporate genetic interactions and rescue experiments of two or more allelic mutations. In our recent study, the genetic interaction between Irf6 and Twist1 causes severe mandible abnormality and cleft of the secondary palate in the mouse model (Fakhouri et al., 2017).

Various studies have used in vivo experiments to test for the expression of various genes involved in craniofacial development. The presence of TGF-β subtypes was studied by using an ex vivo culture model in a serumless, chemically defined medium during mandibular morphogenesis (Chai et al., 1994). Similarly, a study used Alcian blue staining of cultured mandible explants to examine Meckel’s cartilage during morphogenesis in Egfr-/- embryos (Miettinen et al., 1999). A combination of the methodologies from these studies, including an ex vivo mandibular explant described in this report, is useful to characterize the phenotype and signaling pathway in mammalian systems.

Materials and Reagents

  1. 60 mm TC-Treated center-well organ culture dish (Corning, Falcon®, catalog number: 353037 )
  2. Petri dish (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 130182 )
  3. Test tubes (15 and 50 ml) (Denville Scientific, catalog numbers: C1012 and C1062-P , respectively)
  4. Stainless steel grid (0.5 mm hole size) (Home Depot International, model: 3004107 , catalog number: 1001034564)
  5. Millipore type filters (0.8 µm pore size, 47 mm diameter) (Merck, catalog number: AAWP04700 )
  6. Sterile individually packaged pipettes (2, 5, and 10 ml) (Genesee Scientific, catalog numbers: 12-101 , 12-102 , 12-104C )
  7. E10.5-E11.5 murine embryos
  8. 70% ethanol
  9. 95% ethanol
  10. 6-Aminonicotinamide (Alfa Aesar, catalog number: L06692 )
  11. 2 mm-diameter agarose beads or Affi-Gel blue gel (Bio-Rad Laboratories, catalog number: 732-6712 )
  12. Endothelin1 peptide (Enzo Life Sciences, catalog number: ALX-155-001-PC01 )
  13. Thymol (Sigma-Aldrich, catalog number: T0501 )
  14. Sodium phosphate dibasic heptahydrate (Na2HPO4·7H2O) (Sigma-Aldrich, catalog number: S9390 )
  15. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
  16. Potassium chloride (KCl) (Fisher Scientific, catalog number: BP366 )
  17. Potassium phosphate monobasic (KH2PO4) (Fisher Scientific, catalog number: P286-1 )
  18. BGJb medium with L-glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 12591038 )
  19. Fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F0926 )
  20. Ascorbic acid (Fisher Scientific, catalog number: S25184 )
  21. Penicillin-streptomycin 100x (Caisson Laboratories, catalog number: PSL01-100ML )
  22. MEM non-essential amino acid solution 100x (Sigma-Aldrich, catalog number: M7145 )
  23. Glacial acetic acid (Fisher Scientific, catalog number: S25118A )
  24. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P1767 )
  25. Alcian Blue 8GX (National Diagnostics, catalog number: HS-504 )
  26. Alizarin Red S (Acros Organics, catalog number: 400480250 )
  27. Glycerol (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 17904 )
  28. Nuclear fast red (Acros Organics, catalog number: 211980050 )
  29. 10x phosphate-buffered saline (PBS) (see Recipes)
  30. Mandibular explant media (see Recipes)
  31. 3% acetic acid solution (see Recipes)
  32. 2% KOH solution (see Recipes)
  33. Alcian blue solution pH 2.5 (see Recipes)
  34. Alizarin red solution (see Recipes)
  35. 1% KOH / 20% glycerol solution (see Recipes)

Equipment

  1. Surgical scissors (World Precision Instruments, catalog number: 501225 )
  2. Sterile cell culture hood (NuAire, model: NU-545 )
  3. Stereomicroscope (Nikon Instruments, model: SMZ800N )
  4. Forceps (World Precision Instruments, catalog number: 15915 )
  5. Autoflow IR Direct Heat CO2 incubator (NuAire, model: NU-5510 )
  6. Ceramic hot plate (VWR, catalog number: 97042-602 )
  7. Pipette pump (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 9511 )
  8. Autoclave (Future Health Concepts, Primus, catalog number: PRPSS8-A )

Software

  1. NIS Elements AR software (Nikon Instruments) used for the stereomicroscope

Procedure

  1. Embryos (E10.5-E11.5) extraction
    1. Perform euthanization via CO2 followed by cervical dislocation on a pregnant female mouse (see Note 1). All animal procedures were approved by the Animal Welfare Committee (AWC-16-0068) at the University of Texas Health Science Center at Houston and followed the National Institute of Health guidelines as described by Metwalli et al., 2017.
    2. Transfer the animal to a clean procedural room.
    3. Spray ethanol (70%) on the abdominal area of the female.
    4. Use scissors to cut the abdominal skin from the lower abdomen to the epigastric region. Perform the cut at the midline of the abdomen.
    5. Pull the uterus out of the body and cut the connective tissue. This will separate the uterus from the fallopian tubes and ovaries.
    6. Keep all embryos together in the uterus sack.
    7. Place the sack in a Petri dish with cold 1x PBS (15-20 ml, see Recipe 1).
    8. In a sterile hood, separate the embryos by cutting the uterus between the embryos.
    9. Transfer all embryos to a new Petri dish with 1x PBS (15-20 ml) at RT to wash and remove excess blood.
    10. Open the amniotic sac and separate the placenta from the embryos, severing the umbilical cord near the embryo.
    11. Place the embryo in a 15 ml test tube.
    12. Wash each embryo twice with 5 ml of 1x PBS (see Note 2).

  2. Mandibular extraction
    1. In a sterile cell culture hood, dissect the mandibular processes of the first pharyngeal arch under a stereomicroscope while in BGJb culture medium at RT.
    2. Pre-warm BGJb culture medium and mandibular explant media (see Recipe 2) at 37 °C. Add 1-2 ml of PBS at RT to the exterior portion of the center-well organ dish and 0.5 ml of the mandibular explant media to the center-well of the organ dish.
    3. Orient the embryos in a supine position. Using a scalpel, cut through the labial commissure of the mouth at each corner of the mouth in order to separate the mandibular process from the head of the embryo (see Figure 1).


      Figure 1. A murine embryo at E11.5 used for dissecting the mandibular process for ex-vivo assay. The first cut was done at the commissure of the mouth all the way to the hind-brain (A). The second cut was performed at the top of the neck to separate the mandibular process from the rest of the body (B).

    4. Make another cut through each embryo’s neck using a scalpel to completely separate the mandibular process from the rest of the embryo.
    5. Place a single stainless steel mesh across the center-well organ dish, so it is submerged in the mandibular explant media (see Figure 2 and see Note 3).


      Figure 2. Design of mandibular explant on organ-well culture plate

    6. Lay filter paper on top of the metal mesh sheet and allow the filter paper to be submerged in the mandibular explant media (see Note 4).
    7. Place explants in the center of the filter paper.
    8. Place the Petri dish in a CO2 incubator at 37 °C.
    9. Replace the media (0.5 ml) in the center-well with pre-warmed (37 °C) fresh mandibular explant media every 12 h for the first 24 h and then once a day after the first 24 h.
    10. After 12 h of incubation, treat the mandibular sections with 100 mg/ml of 6-aminonicotinamide in mandibular explant media for 3 h. In our study, the 6-aminonicotinamide treatment was used in the experimental samples as a teratogen to the explant (see Note 5) in order to disturb proper tissue development.
    11. After 3 h treatment, replace the media containing the teratogen with fresh pre-warmed (37 °C) mandibular explant media.
    12. In our case, after the teratogen treatment, the experimental mandible samples were treated with 2 mm-diameter agarose beads containing 0.1 μg/ml of endothelin-1 (Edn1) peptide for 8 h in the CO2 incubator at 37 °C (see Note 6). The Edn1 was applied to test whether Meckel’s cartilage and the surrounding tissues are responsive to this signaling molecule and if it would rescue the phenotype of genetic mutations.
    13. After the 8 h treatment, remove the media and the agarose beads that contained Edn1 peptide.
    14. Replace the media (0.5 ml) in the center-well with pre-warmed (37 °C) fresh mandibular explant media. After the first 24 h, replace the media once a day (see Note 7). 
    15. After 24-120 h, fix explants with 95% ethanol for 1-2 days at RT.
    16. Replace the 95% ethanol with 2% KOH (see Recipe 4) for 2-4 h until the explant tissue becomes transparent.
    17. Replace 2% KOH with Alcian Blue staining overnight at RT (see Recipe 5). Alcian Blue stains for acidic glycosaminoglycans found in cartilage.
    18. Replace staining with 95% ethanol for 12-24 h at RT.
    19. Replace the 95% ethanol with Alizarin Red staining (see Recipe 6) for 6 h (Figure 3) at RT. Alizarin Red will stain for calcium containing osteocytes.
    20. Submerge explants in 20% glycerol and 1% KOH (see Recipe 7) to remove excess staining.
    21. Replace solution with 100% glycerol to store at 4 °C. An antifungal such as Thymol may be added to the storing solution (optional).


      Figure 3. Ex-vivo mandibular explant models at E11.5. A. Embryonic mandibular explants extracted at E11.5 and cultured for two days. The image was taken from a side view. B. Alcian Blue stained mandibular explants for Meckel’s cartilage two days post-incubation. The image was taken from the top view of the mandible. C. Alcian blue and Alizarin red staining of mandibular explants after 4 days of incubation.

Data analysis

Skeletal staining was performed on the explants and observed through the stereomicroscope using the software NIS Elements AR. For our study, we examined changes in cartilage and bone formation. In this case, we observed variations including a lack of symmetry, arrested growth of the cartilage, and the shape and thickness of the Meckel’s cartilage. It is recommended to use at least five technical replicates, meaning five embryos from the same litter, for each experimental treatment to account for any intrinsic variation.

Notes

  1. The age of embryos desired to be extracted can be determined by the date of breeding with male and the presence of a copulation plug in the pregnant female.
  2. Each embryo should be washed at least twice. If blood remains, the embryos should be washed until all the blood is removed. The embryos must remain wet during all manipulation steps.
  3. The steel mesh was cut into 1 cm by 1 cm triangles by a guillotine in order to be placed on top of the media in the inner well of the organ culture dish. In our case, the steel mesh was sterilized by dousing it in ethanol and placing it on a fire.
  4. The Millipore filter paper was cut into 1 cm squares, in order to fit within the inner well of the organ culture dish.
  5. 6-Aminonicotinamide was directly added to the medium prior to treatment with Edn1 ligand. Explants without pre-treatment of 6-aminonicotinamide and only treated with Edn1 were included as controls.
  6. Edn1 was delivered by direct pipetting on 2-mm agarose beads and incubated for 5 min. The beads containing Edn1 were placed on either side of the mandibular explants by forceps, and left for 8 h in order for the molecules to diffuse out.
  7. The mandibular explants may be kept in culture for up to one week without any noticeable necrosis or deterioration.
  8. All the tools and solutions used in this protocol should be sterile.

Recipes

  1. 10x phosphate-buffered saline (PBS)
    25.6 g Na2HPO4·7H2O
    80 g NaCl
    2 g KCl
    2 g KH2PO4
    Bring to 1 L with distilled H2O and dissolve the salts by using the ceramic plate and magnetic stir bar
    For 1x PBS, dilute 100 ml of 10x PBS in 900 ml of distilled H2O. Sterilize by autoclaving
  2. Mandibular explant media
    47.5 ml of BGJb medium supplemented with L-glutamine
    3% FBS (1.5 ml/50 ml medium)
    7 mg of ascorbic acid
    0.5 ml of streptomycin-penicillin solution (100x)
    0.5 ml of MEM non-essential amino acid solution (100x)
  3. 3% acetic acid solution
    3 ml glacial acetic acid
    97 ml distilled water
  4. 2% potassium hydroxide (KOH) solution
    2 g potassium hydroxide
    100 ml distilled water
    Mix well by using the ceramic plate and the magnetic stir bar
  5. Alcian blue solution (pH 2.5)
    1 g Alcian blue, 8GX
    100 ml 3% acetic acid solution
    Mix well by using the ceramic plate and magnetic stir bar. Adjust pH to 2.5 using acetic acid
  6. Alizarin red solution
    0.5 mg Alizarin red S
    100 ml 1% potassium hydroxide
    Mix well by using the ceramic plate and magnetic stir bar. Final concentration is 0.005% (w/v) alizarin red in 1% potassium hydroxide
  7. 1% KOH/20% glycerol solution
    20 ml glycerol
    50 ml KOH (2%)
    30 ml distilled water
    Mix well by using the ceramic plate and magnetic stir bar

Acknowledgments

We would like to thank Ali Naji and Dr. Katherine Kin for their excellent help with the ex-vivo mandibular explant assay. The contribution of Victoria Xie who created illustrative Figure 2 is greatly appreciated. This study was supported by the Bone Disease Program of Texas and NIH/R15GM122030-01 to Dr. Walid Fakhouri from the University of Texas Health Science Center, School of Dentistry at Houston. The authors declare no conflicts of interest or competing interests.

References

  1. Chai, Y., Mah, A., Crohin, C., Groff, S., Bringas, P., Jr., Le, T., Santos, V. and Slavkin, H. C. (1994). Specific transforming growth factor-β subtypes regulate embryonic mouse Meckel's cartilage and tooth development. Dev Biol 162(1): 85-103.
  2. Fakhouri, W. D., Metwalli, K., Naji, A., Bakhiet, S., Quispe-Salcedo, A., Nitschke, L., Kousa, Y. A. and Schutte, B. C. (2017). Intercellular genetic interaction between Irf6 and Twist1 during craniofacial development. Sci Rep 7(1): 7129.
  3. Jin, Y. R., Turcotte, T. J., Crocker, A. L., Han, X. H. and Yoon, J. K. (2011). The canonical Wnt signaling activator, R-spondin2, regulates craniofacial patterning and morphogenesis within the branchial arch through ectodermal-mesenchymal interaction. Dev Biol 352(1): 1-13.
  4. Metwalli, K. A., Do, M. A., Nguyen, K., Mallick, S., Kin, K., Farokhnia, N., Jun, G. and Fakhouri, W. D. (2017). Interferon Regulatory Factor 6 is necessary for salivary gland and pancreas development. J Dent Res 1-11.
  5. Miettinen, P. J., Chin, J. R., Shum, L., Slavkin, H. C., Shuler, C. F., Derynck, R. and Werb, Z. (1999). Epidermal growth factor receptor function is necessary for normal craniofacial development and palate closure. Nature Genetics 22(1): 69-73.

简介

发育生物学中的一个主要问题是确定胚胎组织如何及时响应分子信号,并且在形态发生期间给予位置限制指示,其中Meckel软骨是一个经典的例子。 体外外植体模型是一种实用且便利的系统,其允许以受控的方式调查对特定刺激的骨和软骨反应,所述方式严格模拟体内条件。 在这个协议中,外植模型被用来测试Meckel的软骨和周围组织是否对内皮素1(Edn1)信号分子有反应,以及是否会拯救基因突变的表型。 该系统允许在添加到外植体中的外源性化合物的浓度,关于测量下颌骨发育的时间点以及施用外源分子和致畸剂的方法方面进行高度的操作。

【背景】颅面畸形是人类最常见的先天性出生缺陷(Miettinen等人,1999)。许多这些畸形发生在面部形态发生期间,这是一个复杂的多步骤过程,其中颅神经嵴细胞迁移到咽弓以产生许多面部结构(Jin等人,2011)。

特定基因内的两种变异以及基因 - 基因相互作用都可能导致颅面变形。 IRF6基因突变导致头面部外胚层和上皮细胞的形成,导致唇裂,腭裂和下颌畸形。与 IRF6 相比, TWIST1 基因调节胚胎发育期间的神经管闭合和颅骨发育期间的颅缝融合。 TWIST1 中的突变可导致颅缝早闭,下颌发育不全和腭裂(Fakhouri等人,2017)。可以类似于由Miettinen等人(1999)用EGF实施的方法来对IRF6和TWIST1表达进行抑制或改变,使用EGF 受体,以检查其在颅面发育中的作用。然而,当研究开始结合两个或更多等位基因突变的遗传相互作用和拯救实验时,在体内实验中出现困难。在我们最近的研究中,Irf6和Twist1之间的遗传相互作用导致严重的下颌骨异常和小鼠模型中的次级腭裂(Fakhouri等人, 2017)。

各种研究已经用体内实验来测试涉及颅面发育的各种基因的表达。通过在下颌形态发生期间在无血清,化学上确定的培养基中使用离体培养模型研究TGF-β亚型的存在(Chai等人,1994)。类似地,一项研究使用培养的下颌骨外植体的阿尔新蓝染色来检查胚胎(Miettinen等人, ,1999)。这些研究中的方法学的组合,包括本报告中描述的体外下颌外植体,对于表征哺乳动物系统中的表型和信号传导途径是有用的。

关键字:下颌的体外分离, 颅颌面发育, 下颌发育不良, 下颌发育不全, 无颌畸形, 下颌形态, 麦克尔软骨, 胚胎发育

材料和试剂

  1. 60毫米TC处理的中心器官培养皿(Corning,Falcon ,产品目录号:353037)
  2. 培养皿(Thermo Fisher Scientific,Thermo Scientific TM,目录号:130182)
  3. 试管(15和50毫升)(Denville Scientific,产品编号分别为C1012和C1062-P)
  4. 不锈钢网格(0.5毫米孔径)(Home Depot International,型号:3004107,目录号:1001034564)
  5. Millipore型过滤器(0.8μm孔径,47 mm直径)(Merck,目录号:AAWP04700)
  6. 无菌独立包装的移液管(2,5和10ml)(Genesee Scientific,目录号:12-101,12-102,12-104C)
  7. E10.5-E11.5小鼠胚胎
  8. 70%乙醇
  9. 95%乙醇
  10. 6-氨基烟酰胺(Alfa Aesar,目录号:L06692)
  11. 2mm直径的琼脂糖珠或Affi-Gel蓝色凝胶(Bio-Rad Laboratories,目录号:732-6712)
  12. 内皮素1肽(Enzo Life Sciences,目录号:ALX-155-001-PC01)
  13. 百里酚(Sigma-Aldrich,目录号:T0501)
  14. 磷酸氢二钠七水合物(Na 2 HPO 4•7H 2 O)(Sigma-Aldrich,目录号:S9390)
  15. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S7653)
  16. 氯化钾(KCl)(Fisher Scientific,目录号:BP366)
  17. 磷酸二氢钾(KH 2 PO 4)(Fisher Scientific,目录号:P286-1)
  18. 含有L-谷氨酰胺的BGJb培养基(Thermo Fisher Scientific,Gibco TM,目录号:12591038)
  19. 胎牛血清(FBS)(Sigma-Aldrich,目录号:F0926)
  20. 抗坏血酸(Fisher Scientific,目录号:S25184)
  21. 青霉素 - 链霉素100x(Caisson Laboratories,目录号:PSL01-100ML)
  22. MEM非必需氨基酸溶液100x(Sigma-Aldrich,目录号:M7145)
  23. 冰醋酸(Fisher Scientific,目录号:S25118A)
  24. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:P1767)
  25. Alcian Blue 8GX(National Diagnostics,产品目录号:HS-504)
  26. 茜素红S(Acros Organics,目录号:400480250)
  27. 甘油(Thermo Fisher Scientific,Thermo Scientific TM,目录号:17904)
  28. 核固红(Acros Organics,目录号:211980050)
  29. 10倍磷酸盐缓冲盐水(PBS)(见食谱)
  30. 下颌外植体媒介(见食谱)
  31. 3%醋酸溶液(见食谱)
  32. 2%的KOH溶液(见食谱)
  33. 阿尔新蓝溶液pH 2.5(见食谱)
  34. 茜素红溶液(见食谱)
  35. 1%KOH / 20%甘油溶液(见食谱)

设备

  1. 手术剪(世界精密仪器公司,目录号:501225)
  2. 无菌细胞培养罩(NuAire,型号:NU-545)
  3. 立体显微镜(尼康公司,型号:SMZ800N)
  4. 镊子(世界精密仪器,目录号码:15915)
  5. 自动流动IR直接加热CO 2培养箱(NuAire,型号:NU-5510)
  6. 陶瓷热板(VWR,目录号:97042-602)
  7. 移液泵(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:9511)
  8. 高压灭菌器(Future Health Concepts,Primus,目录号:PRPSS8-A)

软件

  1. 用于立体显微镜的NIS Elements AR软件(Nikon Instruments)

程序

  1. 胚胎(E10.5-E11.5)提取
    1. 通过CO 2 2执行安乐死,然后在怀孕的雌性小鼠上颈椎脱臼(参见注释1)。所有的动物程序均由休斯顿得克萨斯大学健康科学中心的动物福利委员会(AWC-16-0068)批准,并遵循Metwalli等人所述的国家卫生研究院指导方针,
    2. 将动物转移到干净的手术室。
    3. 在女性的腹部喷洒乙醇(70%)。
    4. 用剪刀将腹部皮肤从下腹部切到上腹部。
      在腹部中线进行切割
    5. 将子宫拉出体外,切开结缔组织。这将分离子宫从输卵管和卵巢。
    6. 将所有的胚胎放在子宫袋里。
    7. 将麻袋放入培养皿中,用冷的1x PBS(15-20毫升,见方法1)。
    8. 在无菌罩中,通过切割胚胎之间的子宫分开胚胎。
    9. 将所有胚胎转移到一个新的培养皿中,用1x PBS(15-20 ml)在室温下清洗并去除多余的血液。
    10. 打开羊膜囊,将胎盘与胚胎分离,切断胚胎附近的脐带。
    11. 将胚胎放入15毫升的试管中。
    12. 每个胚胎用5毫升的1x PBS洗两次(见注2)。

  2. 下颌提取
    1. 在无菌细胞培养罩中,在RT下在BGJb培养基中,在立体显微镜下解剖第一咽弓的下颌骨过程。
    2. 预温BGJb培养基和下颌外植体培养基(见方案2)在37℃。在室温下将1-2ml的PBS加到中心器官培养皿的外部,并将0.5ml的下颌外植体培养基加到器官培养皿的中心孔。
    3. 将胚胎定位于仰卧位。使用手术刀,切口的每个角嘴唇连合处,以便从胚胎的头部分离下颌骨的过程(见图1)。


      图1. E11.5中的小鼠胚胎用于解剖用于体外实验的下颌骨过程。 (A)第一次切口是在口腔一直到后脑(A)处进行的。第二次切割是在脖子顶部进行的,以便将下颚过程与身体的其余部分分开(B)。

    4. 用手术刀再次切割每个胚胎的颈部,将下颌骨与胚胎的其余部分完全分开。
    5. 将一个不锈钢网放置在中心器官培养皿中,以便将其浸入下颌外植体培养基中(见图2,参见注释3)。


      图2.器官培养皿上下颌外植体的设计

    6. 将滤纸铺在金属网片的上面,让滤纸浸没在下颌外植体中(见注4)。
    7. 将外植体放在滤纸的中央。
    8. 将培养皿置于37℃的CO 2培养箱中。
    9. 每24小时,每12小时更换一次培养基(0.5毫升),预热(37℃)新鲜下颌外植体培养基,最初24小时后每天一次。
    10. 温育12小时后,在下颌骨外植体培养基中用100mg / ml的6-氨基烟酰胺处理下颌骨切片3小时。在我们的研究中,6-氨基烟酰胺处理被用于实验样品中作为外植体的致畸剂(参见注释5)以扰乱适当的组织发育。
    11. 治疗3小时后,用新鲜的预热(37℃)下颌外植体培养基替换含有致畸原的培养基。
    12. 在我们的例子中,在畸胎治疗后,将实验性下颌骨样品在CO 2培养箱中用含0.1μg/ ml内皮素-1(Edn1)肽的2mm直径琼脂糖珠处理8小时在37°C(见注6)。应用Edn1检测Meckel的软骨和周围组织是否对这种信号分子有反应,是否能拯救基因突变的表型。
    13. 8小时处理后,取出含有Edn1肽的培养基和琼脂糖珠。
    14. 用预热的(37℃)新鲜下颌外植体培养基更换中心孔中的培养基(0.5ml)。在第一个24小时后,每天更换一次媒体(见注7)。 
    15. 24-120小时后,在室温下用95%乙醇固定外植物1-2天。
    16. 用2%KOH代替95%乙醇(见方法4)2-4小时,直到外植体组织变得透明。
    17. 在RT下用Alcian Blue染色代替2%KOH过夜(参见配方5)。阿尔新蓝染色软骨中发现酸性糖胺聚糖。

    18. 用95%乙醇替换染色12-24小时
    19. 在室温下用茜素红染色(参见配方6)替换95%乙醇6小时(图3)。茜素红染色含钙骨细胞。
    20. 将外植体浸没在20%甘油和1%KOH中(参见配方7)以除去多余的染色。
    21. 用100%甘油代替溶液,在4°C储存。
      可以在储存溶液(可选)中加入抗真菌剂如百里酚

      在E11.5处,离体下颌外植体模型A.在E11.5处提取胚胎下颌骨外植体并培养两天。图像是从侧面看。 B.培养2天后,阿辛蓝染色的下颌骨外植体用于Meckel's软骨。图像从下颌骨的顶视图被采取了。 C.培养4天后下颌外植体的阿尔新蓝和茜素红染色。

数据分析

在外植体上进行骨骼染色并使用软件NIS Elements AR通过立体显微镜观察。对于我们的研究,我们检查了软骨和骨形成的变化。在这种情况下,我们观察到的变化包括缺乏对称性,软骨的生长停滞,以及麦克尔软骨的形状和厚度。建议使用至少五个技术重复,即来自同一垃圾的五个胚胎,为每个实验处理,以解释任何内在的变化。

笔记

  1. 想要提取的胚胎的年龄可以通过与男性一起繁殖的日期以及怀孕女性中存在的交尾塞来确定。
  2. 每个胚胎应至少洗两次。如果血液仍然存在,胚胎应该被清洗直到所有的血液被移除。在所有操作步骤中胚胎必须保持湿润。
  3. 将钢网用铡刀切成1厘米×1厘米的三角形,以置于器官培养皿的内部培养基的顶部。在我们的案例中,钢丝网是通过将其浸入乙醇中并将其放在火上进行消毒的。
  4. 将Millipore滤纸切成1cm的正方形,以适应器官培养皿的内部孔。
  5. 在用Edn1配体处理之前,将6-氨基烟酰胺直接加入到培养基中。包括没有预先处理6-氨基烟酰胺且仅用Edn1处理的外植体作为对照。
  6. Edn1通过在2mm琼脂糖珠上直接移液并孵育5分钟来递送。将包含Edn1的珠子用钳子置于下颌外植体的任一侧,并放置8小时以使分子扩散出来。
  7. 下颌骨外植体可以保持培养长达一周,没有任何明显的坏死或恶化。
  8. 本协议中使用的所有工具和解决方案应该是无菌的。

食谱

  1. 10倍磷酸盐缓冲盐水(PBS)
    25.6克Na 2 HPO 4•7H 2 O
    80克NaCl
    2克KCl
    2克KH 2 PO 4 4克/克 加蒸馏水至1L,用陶瓷板和磁力搅拌棒溶解盐。
    对于1x PBS,将900ml蒸馏过的H 2 O中的100ml 10x PBS稀释。通过高压灭菌来消毒
  2. 下颌外植体媒体
    47.5ml补充有L-谷氨酰胺的BGJb培养基 3%FBS(1.5ml / 50ml培养基)
    7毫克的抗坏血酸
    0.5 ml链霉素 - 青霉素溶液(100x)
    MEM 0.5ml MEM非必需氨基酸溶液(100x)
  3. 3%醋酸溶液
    3毫升冰醋酸
    97毫升蒸馏水
  4. 2%氢氧化钾(KOH)溶液
    2克氢氧化钾
    100毫升蒸馏水

    使用陶瓷板和磁力搅拌棒充分混合
  5. 阿尔新蓝溶液(pH 2.5)
    1克Alcian蓝,8GX
    100毫升3%醋酸溶液
    用陶瓷板和磁力搅拌棒充分混合。使用醋酸将pH值调整到2.5。
  6. 茜素红溶液
    0.5毫克茜素红S
    100毫升1%氢氧化钾
    用陶瓷板和磁力搅拌棒充分混合。最终浓度为1%氢氧化钾中0.005%(w / v)茜素红
  7. 1%KOH / 20%甘油溶液
    20毫升甘油
    50毫升KOH(2%)
    30毫升蒸馏水

    使用陶瓷板和磁力搅拌棒充分混合

致谢

我们要感谢Ali Naji和Katherine Kin博士对于离体下颌外植体检测的出色帮助。创造说明性图2的维多利亚谢的贡献非常感谢。本研究得到了得克萨斯州骨病研究计划的支持,并得到了得克萨斯大学健康科学中心休斯敦分校牙医学院的Walid Fakhouri博士的支持。作者声明不存在利益冲突或利益冲突。

参考

  1. Chai,Y.,Mah,A.,Crohin,C.,Groff,S.,Bringas,P.,Jr.,Le,T.,Santos,V.和Slavkin,H.C。(1994)。 具体的转化生长因子β亚型调节胚胎小鼠美克尔的软骨和牙齿发育 Dev Biol 162(1):85-103。
  2. Fakhouri,W. D.,Metwalli,K.,Naji,A.,Bakhiet,S.,Quispe-Salcedo,A.,Nitschke,L.,Kousa,Y.A。和Schutte,B.C。(2017)。 Irf6与Twist1在颅面发育过程中的细胞间遗传相互作用 / em> 7(1):7129。
  3. Jin,Y.R.,Turcotte,T.J.,Crocker,A.L.,Han,X.H。和Yoon,J.K。(2011)。 经典的Wnt信号激活剂R-spondin2通过外胚层调节鳃弓内的颅面模式和形态发生-mesenchymal interaction。 开发生物学 352(1):1-13。
  4. Metwalli,K.A.,Do,M.A.,Nguyen,K.,Mallick,S.,Kin,K.,Farokhnia,N.,Jun,G。和Fakhouri,W.D。(2017)。 干扰素调节因子6是唾液腺和胰腺发育所必需的。 J Dent Res 1-11。
  5. Miettinen,P.J.,Chin,J.R.,Shum,L.,Slavkin,H.C.,Shuler,C.F.,Derynck,R。和Werb,Z。(1999)。 表皮生长因子受体功能是正常颅面发育和腭闭合所必需的。 > Nature Genetics 22(1):69-73。
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引用:Jiang, J., Bertol, J. W. and Fakhouri, W. D. (2017). Mandibular Explant Assay for Investigating Extrinsic Stimuli on Bone and Cartilage Development. Bio-protocol 7(23): e2641. DOI: 10.21769/BioProtoc.2641.
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